Liquid-Liquid Phase Separations in Urate Oxidase/PEG Mixtures: Characterization and Implications for Protein Crystallization.
In a previous paper (Vivarès, D.; Bonneté, F. Acta Crystallogr., Sect. D 2002, 58, 472), protein-protein interactions of Aspergillus flavus urate oxidase (Uox) in solution were determined by small-angle X-ray scattering in the presence of different poly(ethylene glycol)s (PEG) in order to correlate second virial coefficient measurements with crystallization conditions. In this paper, we have characterized the experimental phase diagram of urate oxidase in the case of PEG 8000 by determining the solubility curve and the dilute part of the liquid-liquid phase separation (LLPS). Within this phase diagram, different mechanisms of urate oxidase crystal growth and LLPS can be observed by optical video microscopy. The influence of the LLPS on both the mechanisms and kinetics of urate oxidase crystal growth was observed by optical microscopy and small-angle X-ray scattering (SAXS). Interactions between the macromolecules were studied by SAXS in the dilute and dense phases of the demixed solution. It was observed that the LLPS precedes and slows down the crystallization. This study shows that urate oxidase is a good model to study protein/PEG mixtures in the general context of protein crystallization.
Vivarès
D
D
CRMCN-CNRS, Campus de Luminy, Case 913, F-13288 Marseille Cedex 09, France, and LMCP-UMR7590, Case 115, 4 place Jussieu, F-75252 Paris Cedex 05, France.
Bonneté
F
F